Clostridium difficile is a leading cause of nosocomial infections worldwide. Despite efforts to improve detection and limit transmission of the bacteria, incidence rates continue to rise. This rise is primarily due to the overuse of broad-spectrum antibiotics, which disrupt the normal, healthy gut microbiota and the natural colonization resistance that it provides. Current C. difficile infection (CDI) therapies are effective at reducig pathogen shedding and elimination of symptoms associated with acute C. difficile associated disease (CDAD); however, rates of relapse and recurrence after an initial CDI are still high, possibly due to the collateral damage induced to the otherwise protective healthy microbiota. Therefore, there is a clear, unmet medical need for a safe and effective anti-bacterial agent that can prevent or treat CDI relapse and reinfection after a cured initial episode.? AvidBiotics has developed specific protein platforms capable of producing potent, highly-targeted bactericidal protein complexes that are well-suited for prophylaxis and preventing bacterial infections. Each platform employs a bacteriocin protein assembly analogous to the contractile tail structures of Myoviridae bacteriophages. They rapidly and specifically kill target cells by binding to a surface molecule via receptor binding proteins (RBP) on their tail fibers, followed by bactericidal puncture of the cell envelope. We have demonstrated the tailorability of this platform by fusing the bacteriocin tail fibers to the RBP containing tail fibers of bacteriophages, thus engineering novel bacteriocins (Avidocins(tm)) against a wide variety of bacteria. This strategy has produced more than 15 highly specific Avidocin-CD products. Just 6 are needed to target strains expressing 10 of the 12 known allelic variants of slpA present in the C. difficile population. In pre-clinical mouse studies Av-CD291.2, a potent Avidocin-CD targeting allelic variant 4, did not disrupt healthy gut microbiota or the colonization resistance it provide and demonstrated efficacy in preventing antibiotic-induced colonization by spores from a hypervirulent, riboptype 027 strain. While a cocktail of 6-7 Avidocin-CDs that covers >90% of the clinically relevant N. America strain types is possible, it would not be practical. Therefore, e are proposing to identify new phage RBPs, or modify existing ones, to construct individual Avidocin-CDs (or a cocktail of 2-3) with coverage of ?80% of relevant C. difficile strains. The R21 Phase will be devoted to the construction and pre-clinical testing of a new generation of Avidocin- CD(s) with ?broader C. difficile coverage than the products currently constructed. ?After optimizing Avidocin-CD delivery formulation for mice, we will determine the pre-clincial impact treatment has on ?colonization resistance as well as their efficacy in preventing C. difficile ?reinfection after vancomycin treatment of a CDI episode.?The purpose o the R33 Phase is to develop a manufacturing process suitable for large-scale Avidocin-CD production and to generate preliminary safety data in an animal model. Data from both activities are expected by the FDA in a pre-IND meeting package.